Transcript RNA Super chip

RNA Switches
Genetic Research Tools
RAVINDER NAGPAL1, MALVIKA MALIK1, MONICA PUNIYA2, AARTI
BHARDWAJ3, SHALINI JAIN4 and HARIOM YADAV4*
1Dairy Microbiology, 2Dairy Cattle Nutrition, 4Animal Biochemistry,
National Dairy Research Institute, Karnal 132001,
Haryana, 3 Meerut Institute of Engineering and Technology, Meerut-250002,
U.P., India.
Email: [email protected]
Role of RNA inside the Cell
• According to traditional central dogma of
genetics RNA is the intermediate carrier of
genetic information between DNA and
Protein.
DNA
Transcription
RNA
Translation
PROTEIN
New roles discovered for RNA
• Certain RNA molecules, ribozymes, could catalyze
biochemical reactions, a job previously thought to be the
exclusive province of enzymes, which are proteins.
(Kruger et al, 1980)
• Certain RNA molecules could behave like another type
of protein: antibodies. Researchers synthesized RNA
molecules - now known as aptamers- that, like
antibodies, can latch on tightly to specific target
molecules.
(Ellington and Szostak, 1990)
contd...
• Certain RNA sequences can directly sense
environmental factors and small molecule metabolites,
this allow the associated mRNA to regulate their own
transcription or translation.
(Breaker, 2002)
• These self regulatory messages are called as RNA
sensors and RNA switches.
RNA Switches
• RNA switches are mRNAs that sense the environment
directly, shutting themselves down in response to
particular chemical clues.
• Breaker, Nudler, Yura and Cossart laboratories report
that specific RNA sequences can act as environmental
sensors of vitamin cofactors (including vitamins B1, B2
and B12) and temperature, which allow them to directly
regulate the transcription or translation of associated
mRNAs.
contd...
• Distinct RNA molecules directly perform or mediate
enzymatic processes such as RNA cleavage, splicing
and translation.
• Non-coding RNAs are involved in a tremendous
variety of gene regulatory mechanisms that operate at
both the DNA and mRNA level .
• Seven types of RNA switches have been found in
bacteria so far. These include switches controlling the
manufacture of the vitamins B1 and B2 and the
nucleotide guanine.
Mode of action of RNA Switches
• RNA Switches can regulate gene expression at both
transcription as well as translation level.
• At transcription level they can induce premature
termination of mRNA transcript.
• At translation level the initiation of protein synthesis is
stopped by these switches.
• In certain cases RNA switches have self-cleavage
property, thus regulating their own expression.
• RNA switches can also act as thermo sensors,
regulating gene expression according to the
temperature change.
Anti-AntiTerm.
Antiterm.
Term.
RNA poly.
DNA
+ LIGAND
RNA poly.
Regulation by Transcriptional Termination
Examples of Transcription Termination
Mechanism
• Transcription termination mechanism has been reported
functional in the regulation of rib and thi operon in
Bacillus subtilis.
(Mironova et al, 2002)
• rib operon :- riboflavin biosynthesis.
• thi operon :- thiamine biosynthesis.
• In case of rib operon the 5’ Untranslated Region of the
mRNA transcript can fold into two confirmations,
depending upon the presence or absence of FMN.
contd...
• In low concentration/absence of FMN the nascent
transcript come out of RNA polymerase and attain a
confirmation that allow the transcription to be
completed.
• In presence of FMN, it binds to the nascent transcript
and transcript folds in such a way that the
transcription is terminated , resulting in premature
transcript which leads to no gene expression.
Anti-anti-SD
Anti-SD
SD AUG
Ribosome
+LIGAND
Anti-anti-SD
SD
Anti-SD
Regulation by Translational Initiation Inhibition
Example of Translation Inhibition Mechanism
• Translation initiation inhibition mechanism has been
proposed for regulation of thiM and thiC genes
required for biosynthesis of thiamin.
(Mandal and Breaker, 2004)
• The translation initiation for these genes is sensitive to
the presence of thiamin.
• Binding of thiamin to the mRNA precludes the
Ribosome Binding Site from ribosome subunit access.
Observations:
• Gram-positive bacteria are more likely to couple these
elements to a terminator/anti-terminator system, that is
at the level of transcription.
• Gram-negative bacteria more typically link these
elements to a SD-sequestration mechanism, that is at the
level of translation.
contd...
• This type of regulation is found to be economic for the
bacterial system as:– In Gram positive bacteria the genes for one biosynthetic
pathway are arranged in a cluster, therefore they are regulated
at the transcription level.
– In Gram negative bacteria the genes for one biosynthetic
pathway are scattered along the whole genome and are
therefore regulated at the translation level.
Self-cleavage of the RNA molecules
• The regulation of glmS gene(glutamine-fructose-6phosphate amidotransferase)in B. subtilis is by
associated activity of ribozyme and riboswitches.
• In this case the 5’ UTR of glmS binds with gluosamin6-phosphate, product of GlmS activity and start acting
as ribozyme, due to conformational change.
• This results in the self cleavage of the mRNA by an
internal phosphoester transfer.
Secondary Structure of self cleavage RNA molecule
Activation of gene expression by
RNA Thermo sensors
• There are two well-studied examples of
temperature-dependent regulation of gene
expression and/or activity:-
– during the heat shock response
– during pathogenic invasion
• Almost all the known thermo sensors regulate the
gene expression at the translational level.
Translation regulation by an mRNA Thermo sensor
Ribosome
mRNA
Temperature Increase
Examples of RNA Thermo sensors
• Translation of LcrF, a general activator of virulencerelated gene expression in Yersinia pestis, was found to
be thermally regulated.
(Hoe and Goguen, 1993)
• A new report from the Cossart lab (2002) now provides
strong evidence for an RNA thermo sensor that regulates
translation of PrfA, a general activator of virulence
genes in a pathogenic variety of Listeria, L.
monocytogenes.
Regulation of virulence gene in L. monocytogenes
• In L. monocytogenes prf A gene is responsible for the
virulence of the organism.
• The prfA gene is transcribed at both 30°C and 37°C.
• But it is translated only at 37°C.
• sequences in the 5' UTR of the prfA mRNA could
form an extended hairpin that includes the SD
sequence.
• At 37°C, when the pathogen enters into the host
system, this structure is destabilized the SD sequence
is free initiation of translation.
STRUCTURE OF RNA SWITCHES AND THEIR LIGANDS
Natural small molecule-regulated RNA switches
Advantages of RNA switches for the
organisms possessing them
• Stringent control: RNA switches are very specific to
their substrate. eg. recognition of TPP by THI boxes is
1000 times greater than for either thiamine or thiamine
monophosphate.
( Winkler et. al. 2004)
• Ligand can be reused: Binding of the substrate to the
RNA molecule is required for a short time, after this the
ligand can be reused for the same or different purpose.
• Only one gene is involved: There is no involvement of
another gene product, which reduces the effect of
mutation that will deregulate the mechanism.
Gene expression regulation by RNA in Eukaryotes
De-repressor RNA:
– Recent data indicate that
small non-coding RNA
species function as coactivators of eukaryotic
gene transcription .
– Small activator or derepressor RNA molecules
play a role in initiating and
stabilizing transcription
bubbles, mRNA synthesis
by RNA polymerase.
(Frenster, 1965)
dsRNA as a regulator of gene expression
 ds RNA has role in several chromatin and/ or genomic
DNA modifications, which lead in the regulation of
specific genes.
 ds RNA dependent mechanism can act at both
transcriptional as well as post transcriptional levels.
This type of gene expression is given different names in
different organisms.
– RNA interference (RNAi) , in case of animals.
– Post transcriptional gene silencing (PTGS) , in case of
plants.
– Quelling, in case of filamentous fungi.
Other RNAs involved in regulation of gene expression
– micro RNA (mi RNA)- a class of noncoding small
RNA identified for their role in translational
repression in some animals.
– short hairpin RNA (sh RNA)- a special class of RNA
that becomes double stranded by folding of the RNA
strand over itself, has been shown to be responsible
for the transcriptional regulation.
Applications of RNA switches
in genetic research
• RNA switches can be used to create small moleculeregulated transgenes, which may allow researchers to
manipulate expression of any individual construct
within a battery of simultaneously introduced
experimental constructs.
(Werstuck and
Green, 1998)
• We can imagine exploiting RNA thermoregulation to
create heat-inducible transgenes.
(Brand and Perrimon, 1993)
contd...
• Aptamers have been placed within 5' UTRs and shown
to inhibit gene expression upon introduction of the
appropriate ligand.
(Werstuck and Green, 1998)
• Self-cleaving RNAs can be placed under allosteric
control by various small molecules, which can then be
used to analyze the composition of chemical and
biological mixtures .
(Seetharaman et al, 2001)
• RNA switches can be used in gene therapy, allowing
the patient to take pills to switch introduced gene on or
off.
contd...
• It may be possible to design drugs using RNA switches’
cognate ligands that would constitutively repress
associated gene activity.
– Such compounds would efficiently inhibit bacterial
growth by simultaneously repressing multiple
components in a given biosynthetic/metabolic/transport
pathway.
– Such compounds might be likely to have relatively low
toxicity, as they would be designed to target RNA, not
protein.
RNA on a Chip
• In 1995, Breaker successfully engineered RNA-based
molecular switches.
– A molecular switch is a molecule that turns on or off by
another molecule or compound.
• With dozens of these switches on hand, Breaker
created an array of biosensors that use RNA to
measure or detect compounds.
RNA Biochips
An array of RNA molecular switches constructed using seven
distinct effector-modulated ribozymes based on the hammerhead
self-cleaving RNA.
Array for RNA
• Breaker placed the RNA switches on a gold-coated
silicon surface and arranged them in clusters.
• Each switch was designed to bind only to a specific
molecule its “target” and then to release a signal that
identifies the target molecule. (In the prototype, the
switches released a radioactive signal.)
• This array of RNA switches was tested on a variety of
complex mixtures. In one experiment different strains of
E. coli found in bacterial cultures were successfully
identified.
• The array's ability to simultaneously identify a
potentially large number of compounds, combined
with the precise exclusivity of each switch, adds up to
a recipe for a powerful and wide-ranging laboratory
on a dime-sized slice of silicon.
RNA Super chip
• Future RNA chips, capable of revealing the molecular
composition of complex mixtures-like blood serum
and industrial waste-far more comprehensively than
current biochips can be synthesized.
• Advanced versions of RNA biochip could be used
for many different targets like drugs, toxins and
metabolites, as well as proteins and nucleic acids.
Benefit of using RNA biochip
• Benefit of RNA switches is their ability to withstand the
sometimes unpredictable and harsh environment outside
the lab as compared to a protein biochip.
• Breaker's RNA switches have been engineered to refold
back to their original form after heating, this snap-back
character will give RNA biochips a considerable
advantage for use in more exotic test environments.
Obstacles
• Manufacturing costs .
• The chemical stability of the switches .RNA is
vulnerable to certain chemicals often found in
test situations that can disintegrate a switch.
• The finer points of molecular recognition .
• This technology is so new that it is unclear just
how many different compounds it will prove
possible to recognize .
Future Prospects
• RNA switches must be engineered to release a
fluorescent, rather than a radioactive signal.
• Use of an RNA chip in diverse fields like chemical
engineering, environmental science, and even
biological and chemical warfare defense.
• Use of RNA chip in the detection and identification
of pathogens.
Conclusion
• Apart from being the carrier of genetic
information , RNA can also act as the regulator of
expression this information. These regulators are
called as RNA switches.
• RNA switches have been discovered to be
involved in the regulation of gene expression in
prokaryotes.
• RNA switches can be used in genetic research to
study the effect of environmental changes on
particular gene expression.
• RNA switches can be used to in gene therapy and
as drugs.
• Researchers are trying to synthesize artificial RNA
switches and we can hope that in future we will be
able to use RNA switches not only in genetic
research but also in antimicrobial therapy, as
biosensors for different chemicals and in detection
of pathogenic microorganisms.